The origin of black and white coloration of the Asian tiger mosquito Aedes albopictus (Diptera: Culicidae)

Micro- and nanostructures of the white and black scales on the tarsi of the mosquito Aedes albopictus are analysed using scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Reflectance spectra of the white areas are measured. No clear difference is present in the morphology of micro- and nanostructures of black and white scales in SEM and TEM, but black scales contain a dark pigment. The white colour of the scales has a structural origin. The structural white produced by the micro- and nanostructures of the scales on the tarsi of Ae. albopictus appears bright and is angle-dependent, since the reflected light changes according to the angle detection and according to the tarsus orientation. The optical appearance of the scale system of Ae. albopictus has a complex nature and can be explained by the combination of several effects. Among them, multiple refraction and reflection on the micro- and nanostructures of the scales are mainly responsible for the white appearance. The results suggest that mosquito scales, in addition to their superhydrophobic function, produce structural white. The biological role of white and black patches in mate recognition and defensive behaviour in the mosquitoes of the genus Aedes is hypothesized.


Introduction
Body color (coloration) and light signals (bioluminescence) have a fundamental role in insect inter-and intra-specific visual communication allowing for species recognition, mating, prey capture, and predator avoidance [1]. Insect colours may be due to pigments (e.g., melanins, carotenoids, ommochromes, and pteridins situated in the cuticle or under a transparent cuticle) [2] able to absorb visible electromagnetic radiation in a selective way, or due to various physical phenomena, such as reflec-tion, refraction, interference, diffraction and scattering, that cause the selective reflection of light [3]. Quite often, structural colors are present together with pigment colors, to increase or to reduce the brightness and to produce particular effects [4]. Insect exoskeletons with their multilayered internal organisation and the cuticular micro-and nanostructures present on the surface offer remarkable examples of structural colours in the animal kingdom. The relationship between shining (sometimes iridescent) colors and surface microstructures located on scales has been extensively studied in butterflies, especially in the Morpho genus [4][5][6][7].
Among Diptera, representatives from the family Culicidae bear scales typically covering their body [8,9]. Their shape and structure vary among different taxa and have been previously used as a taxonomic character [10]. The function of the body scales in mosquitoes is probably related to the superhydrophobicity of their body surface, as an adaptation of an aquatic insect to the subaerial life at the adult stage. Indeed, leg scales with their nanostructures are able to entrap air [11][12][13] and play an important role in contact with water during egg laying, giving the mosquito high water buoyancy and floating ability, and during emergence from the aquatic pupa, preventing adults from wetting and allowing them to fly away from the water surface without being trapped by capillary forces.
The scales on the mosquito body can also produce a colouration pattern, which is often used in the species determination [14,15]. Notwithstanding such scales are rather similar to those of butterfly wings [16], the mosquito scale nanostructures have not been deeply investigated so far regarding the structural colours they generate. Structural colours are common in insects [4] and have been described mainly in Lepidoptera and Coleoptera [16,17]. As far as Diptera are concerned, investigations on structural colours are scanty.
The aim of the present investigation is to describe in detail the nanostructures and microstructures of the scales in the Asian tiger mosquito Aedes albopictus Skuse (Diptera: Culicidae). The species belonging to this genus (among which Ae. aegypti L. is present) are visually distinctive, because they have noticeable black and white markings on their bodies and their legs, whose different distributions in different species allows for species identification. The ultrastructure of the white and black scales on the hindlegs of Ae. albopictus is analysed using scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Moreover, reflectance spectra of the white areas are measured. The scales are present also on other body parts, but we mainly focused our attention on the hindlegs, because of their bright white stripes.
Ae. albopictus or Asian tiger mosquito is an invasive species native to tropical and subtropical areas of Southeast Asia, nowadays established in all Mediterranean countries. It is recognised as one of the 100 most invasive species in the world being an aggressive day-biting species. It is of high medical importance as a vector of chikungunya virus, dengue virus, and dirofilariasis [18]. Detailed studies regarding the optical properties of the body surface of insects, such as Ae. albopictus can be relevant to both species recognition and surveillance. Moreover, these studies increase our knowledge on inter-and intraspecific communication of these dangerous species, thus helping to develop new control methods.

Results
The body of the adult (male and female) of Ae. albopictus is typically characterised by the presence of white and black areas ( Figure 1). White patches are present on abdominal segments, thorax, head, and legs of both sexes. They are particularly evident on the hindlegs (Figure 1c-f). Males and females differ in size and in shape of antenna and mouthpart, but their hindlegs are similar in colour and shape. Their tarsi are characterised by five segments, whose length decreases from the first to the last tarsal segment. Each segment is white in its proximal portion and black in its distal portion, except for the last segment, which is totally white (Figure 1f).
Observations with SEM reveal that the body of Ae. albopictus is covered with scales and microtrichia ( Figure 2). Scales of different shape are present on different body parts. Spatulate scales are the most common kind of scales. They are found on the thorax (Figure 2a To analyse the micro-and nanostructures located on the scales of Ae. albopictus in detail, we focused on the tarsi of the hindlegs, which show particularly evident white patches. In particular, we examined the scales at the articulation between different tarsal segments of the hindlegs, where black scales are situated very close to white scales (Figure 3a and inset). Scales are spatulate, convex and show a rounded apex. Their size is variable, and some small scales are visible under wider scales ( Figure 3a). The lower surface of a scale is flat, without specialized micro-and nanostructures (Figure 3b). The upper surface is covered by longitudinal ridges (Figure 3c-e). The distance between the ridges is 1.189 ± 0.03 µm (average ± SE) in the black scales and 1.283 ± 0.03 µm in the white scales. The cuticle between longitudinal ridges is decorated with anisotropically situated nanostructures with a herringbone pattern (Figure 3e). The ridges show overlapping lamellae from which fine folds Their lower and the upper surfaces are closely juxtaposed, even if some empty spaces (nanovoids) are occasionally visible between them (Figure 4b-d,h). Such nanovoids originate from the rests of epidermal cells and appear in TEM as white or light grey areas inside the scales, together with electron-dense debris ( Figure 4h). Their occurrence is higher at the bases of microribs, because the cuticle thickness is higher there (Figure 4h). Along the upper surface of the scale, the longitudinal ridges run orthogonal with respect to the herringbone pattern floor (Figure 4b,c,f,g). The cuticular microribs along the ridges appear in the cross section as small lateral globular bulges (Figure 4c,d,f,g). We did not observe any clear difference in the morphology of black and white scales in SEM and TEM, except for the small difference in the distance between the longitudinal ridges.
The scales appear black and white only under reflected light at low magnification. In our observation of the tarsal scales of Ae. aegypti in a light microscope under transmitted and reflected light we could observe that in dry specimens under transmitted light white scales appear transparent and black scales appear dark (Figure 5a,b). Under reflected light, at high magnification, both white and black scales demonstrate a faint iridescent spotted pattern (Figure 5a,b). The colour of the pattern depends on the illumination direction in black scales, while in the white scales only the spot saturation changes at different illumination directions. Scales loose the whitish coloration when observed immersed in oil (with a refractive index closer to that of the cuticle material as to that of air) under    White scales look transparent under these conditions, white black scales remain dark because of melanization. All reflection spectra are homogenous without evident peaks ( Figure 6). The reflection intensity in the near-infrared spectral range (>900 nm) is about 2.5 times stronger than the WS1 reflection (!) and up to five times stronger (at 45° detection) than in the UV spectral range (<300 nm) ( Figure 6). Pronounced specular reflection (at 45° detection) could be seen at proximal and distal illumination ( Figure 6). Enhanced reflection at 30° detection and distal illumination (compared with proximal illumination) (Figure 6a,b) should be related to the tilt angle of the individual scales (Figure 3a). There is also relative strong light scattering on the thin scale surface structure (Figure 3c-e; Figure 4), which amounts for even backwards reflection (proximal illumination, −20° detection) of about 1/3 of the WS1 standard reflection (at 45° illumination/ detection). From geometrical considerations, it is clear that backwards reflection (−20° detection) is the strongest and reflection at 30° is the weakest at perpendicular illumination. In UV spectral range, the light scattering has no specific trends (corresponding to, e.g., Rayleigh scattering), though absorption by molecules with aromatic groups might modulate the reflection. There are no pigments in the white scales (Figure 5d), so interference on the single scales structure is responsible for the reduced visible and UV (compared to near-IR) reflection.
Sputter coating with Au/Pd (10 nm thickness) of the tarsal scales of Ae. albopictus (Figure 7) almost eliminates the visual difference between white and black scales. Since the scale surface is optically smooth, the metal-coated scales demonstrated specular reflection. A thin Au/Pd film on the scale surface effectively screens the pigment presented inside the scales and traps the light so that the white as well as the black appearance of the scales disappears.
Observations of the legs of dry adults using a fluorescence microscope revealed that the white areas on the tarsi show UV-induced fluorescence (Figure 8). The excitation occurs at 365 nm (UV light) and the emission from 397 nm (blue light).

Figure 6:
The light scattering spectra from a white area of the hindleg tarsus of Aedes albopictus male at different leg orientations. The white spot was illuminated at 45°. The light scattering was detected at 45°, 30°, and −20° (relative to the normal to the surface). Illumination was performed at different leg orientation relative to the illumination source: illumination was from the proximal direction (a), from the distal direction (b), and perpendicular to the tarsus main axis (c). Reflection at 45° for different leg orientations is summarized in (d).

Multifunctional role of the scales
The present investigation reveals that the white bright scales on the body of Ae. albopictus are constituted of transparent cuticle, and their micro-and nanostructures can generate structural white. These results suggest a multifunctional role of the mosquito scales which, in addition to a superhydrophobic function [11][12][13], produce structural colours similar to those of butterfly scales. The involvement of these structures in the origin of structural white is clearly demonstrated by our optical microscopy observations. Indeed, when we observed the tarsal scales under transmitted and reflected light, we could observe white bright scales only in dry specimens under reflected light, while the white scales observed under transmitted light appear transparent. Moreover, the white scales immersed in oil with a refractive index close that of chitin do not appear white, but rather transparent, while the black scales appear dark due to the presence of a certain amount of a dark pigment melanin in the cuticle. This means that neither pigments nor inclusions with strongly different refractive index (e.g., voids) are responsible for white appearance of the scales.
Structural whiteness requires scattering processes for all visible wavelengths [19]. Two kinds of structural whiteness can be recognised, namely diffuse whiteness, which is angleindependent and metallic silver, which is angle-dependent. Diffuse whiteness produces matt white and is due to the multiple scattering of light within a randomly structured medium, such as the three-dimensional photonic solid in the scales of Cyphochilus spp. beetles [20]. An example of structural white in Diptera has been recently described in the white patches located on the thorax of Bactrocera oleae (Rossi) (Diptera: Tephritidae) [21]. In this fly, the structural white is due to modified air sacs under transparent cuticle and is independent on the diffuse angle. This structure shows internal arborisations with beads in an empty space, constituting a three-  dimensional photonic solid responsible for light scattering. Metallic silver produces a bright, shining white caused by a variation of the reflected light intensity under different incident or viewing angles, such as in the scales on the ventral side of the wings of the butterfly Curetis acuta Moore (Lepidoptera: Lycaenidae) [22] or in the Ae. albopictus scales described here. Indeed, the structural white produced by the micro-and nanostructures of the scales on the tarsi of Ae. albopictus appears bright and is angle-dependent since the reflected light changes according to the angle detection and according to the tarsus orientation (from the proximal direction, from the distal direction, and perpendicular to the tarsus main axis).

Optical effects on the mosquito leg scale system
The optical appearance of the scale system of Ae. albopictus has a complex nature and can be explained by the combination of several effects, which contribute to different extent: (1) the cylindrical shape of the leg, which may alter scattering depending on the illumination/observation configuration; (2) light scattering, which might be subdivided in (a) Rayleigh scattering on nanostructures, (b) scattering on partially regular micro-and nanostructures, and (c) thin film interference scales; (3) selective absorption by various molecules present in the cuticle; and (4) collective effects of the overlapping scales. The whole structural hierarchy determines the reflection properties of the white scales, but different micro-and nanostructures are presumably involved in the light scattering at different illumination/detection conditions. Below, we provide a detailed discussion of these effects and their contribution to the measured spectra.

Cylindrical shape of the leg
The cylindrical shape of the leg in combination with the structural white makes the white scales well visible, independent on the observation direction in the plane perpendicular to the legs (Figure 6c). The reason for this is that in the plane crossing the cylinder perpendicular to the cylinder axis, a point on the cylinder surface exists where the incident and reflected angles (from the light source and to the detector, respectively) are equal.

Rayleigh scattering
The contribution of the Rayleigh scattering to the white appearance is almost negligible. The scales look slightly bluish under a light microscope, but there is no evidence for a λ −4 -dependence (typical for Rayleigh scattering) in the reflection spectra that increases with increasing wavelength (Figure 6). Besides, the scales are transparent when they are immersed into oil. This means, that there are no or just a few intracuticular nanostructures whose refractive index differs from that of the cuticle itself. In the TEM images, only a small number of nanovoids could be found (Figure 4).

Scattering on micro-and nanostructures
Multiple refraction and reflection on the ridges and microribs, as well as reflection from the upper surface of the scales between ridges (and the corresponding herringbone surface pattern) are mainly responsible for the white appearance of the scales. Coating of scales with a thin Au/Pd layer eliminates the white (and also the black) appearance and demonstrates the importance of multiple reflections for the appearance of the white scales (Figure 6c).

Thin film interference
In the microscopic areas on the scales with relatively constant cuticle thickness, the effect of interference in thin films can be observed, which modulates the reflection spectra. Its contribution is about 11% (considering the refractive index of chitin of 1.56 and an illumination angle of 45°), and it is visible as faint colours on the white background in microscopic observations (Figure 5a). A similar but weaker interference is present in the black scales (Figure 5b) because of the partial light absorption by melanin.

Light absorption
Some substances absorbing UV light are present even in the white scales. They attenuate the reflection in the UV-visible, but not in the red-IR, spectral range. Multiple refractions and reflections enhance this effect.

Collective effects of the overlapping scales
Finally, the fact that the scales overlap each other (Figure 5d) may lead to an increase of both reflection and scattering. Thus, the specular reflection from four overlapping chitin scales at 45° should be three times stronger than the reflection from a single scale. The variation in the scale orientation at the level of one scale and between neighbouring scales is responsible for the strong small-angle scattering and for the absence of the prominent specular reflection. The scales are tilted with respect to the surface and directed distally (Figure 5d). Highly diffuse scattering might be the only reason for the strong reflection at 30° and distal illumination compared to proximal illumination (Figure 6a,b).
Therefore, the main contribution to the white coloration is provided by non-coherent light scattering (diffuse scattering) on hierarchical randomized multiscale 3D structures (Figure 4a-d,  Figure 3d). The coherent contribution (interference), which was estimated to be around 10%, appears due to structure regularity.

Functional significance of the mosquito leg scale system
Structural colors producing matte white and bright silver brilliancy can be readily distinguished and can be involved in interand intraspecific visual communication including recognition and mating [23,24]. Our data on Ae. albopictus scales and the above discussion show that the main contribution to the white appearance of the scale system comes from the multiple refraction and reflection on the ridges and microribs. Interestingly the white scales of Ae. albopictus show UV-induced blue autofluorescence as it has been demonstrated by [24] and confirmed here, exactly as it happens in the white patches of the olive fruit fly B. oleae [21]. The biological role of white patches is not known, neither in the olive fruit fly B. oleae nor in the mosquitoes of the genus Aedes. However, we can hypothesize a role in mate recognition in Ae. albopictus. The use of visual cues in mate recognition has been described in other Diptera, such as the two predatory fly species Lispe consanguinea Loew (Diptera: Muscidae) and L. tentaculata DeGeer (Diptera: Muscidae), which use visual cues from reflective concave silvery scales on the head face during mating [25]. It is well known that in mating swarms, mosquitoes mainly use acoustic signals created by conspecific wingbeats to locate and respond to one another through harmonic convergence [26]. Yet, visual trap components can be important, especially in diurnally active mosquitoes, such as the yellow fever mosquito Ae. aegypti (and the tiger mosquito Ae. albopictus). Indeed, it has been previously demonstrated that the use of visual cues can improve the attractiveness of sound-baited traps [27,28]. The mechanisms underlying swarm formation and long-range detection of females by males remain largely unexplored in mosquitoes of the genus Aedes, but an investigation [29] demonstrated that swarm formation and mate recognition in Ae. aegypti are mediated, in part, by visual cues represented by wingbeat light flashes. Such flashes had no signal function for crepuscular house mosquitoes, such as Culex pipiens L. (Diptera: Culicidae) [29].
Another possible role of the black and white coloration of the Aedes genus could be related to defensive behaviour. The influence of prey coloration of animals living in groups on the "confusion effect" towards predators is a field still largely unexplored. However, there is some evidence in "human predators" that motion dazzle camouflage could enhance the confusion effect [30][31][32]. In this context, fast moving black-and-white mosquitoes reflecting light that changes owing to the angle-dependent structural white produced by the scales could create a visual effect causing confusion in predators and disrupting perception. Further studies are necessary to test this hypothesis.
In conclusion, the present research analysed in detail the ultrastructure of the scales producing bright white areas alternating with dark areas on the tarsi of the mosquito Ae. albopictus. We could identify micro-and nanostructures responsible for the production of an angle-dependent structural white, confirmed by reflection spectra measurements of the white areas. We believe that to deepen the knowledge on the nanostructures producing structural colours in mosquitoes of the genus Aedes, is important not only to widen our knowledge on the origin, variability, and function of structural colours in insects, but also to better understand the nature of the cues potentially driving the behaviour of these medically and economically significant insects. This knowledge will allow for the development of more effective traps to collect, monitor, and control these dangerous insect species.

Experimental Insects
Single alive females of Ae. albopictus were collected in Perugia, Italy in vials using human beings as attractants. Insects were placed in a net cage (300 mm × 300 mm × 300 mm) and transferred to the laboratory. In the laboratory, some specimens were immediately fixed for TEM investigations, while some specimens were frozen at −20 °C for further use. For the terminology regarding the morphological description of mosquito scales we used as reference [16].

Light microscopy
Tarsal black and white scales of Ae. albopictus were observed using light microscopy with transmitted and reflected light in an inverted bright-field microscope ZEISS Axio Observer (Carl Zeiss Microscopy GmbH). Dry specimens and specimens immersed in oil (immersion oil 518, Carl Zeiss, Oberkochen, Germany) were observed. Sputter-coated samples (see SEM procedure) were observed using a stereomicroscope under reflected light.

Fluorescence microscopy
Observations of the legs of dry adults were performed using a fluorescence microscope Zeiss Axiophot (Zeiss, Jena, Germany) with an excitation filter 365 nm, chromatic beam splitter FT 395 nm, emission 397 nm, with an excitation filter 450-490 nm, chromatic beam splitter FT 510 nm, emission 520 nm, and with an excitation filter 546 nm, chromatic beam splitter FT 580 nm, emission 590 nm. Insect legs were removed from the insects and placed on a microscope slide for observations.

Scanning electron microscopy and cryo-SEM
Dry insects were observed in a scanning electron microscope Hitachi S-4800 (Hitachi High-Technologies Corp., Tokyo, Japan) at 3 kV acceleration voltage. Specimens were sputtercoated with gold/palladium (10 nm layer thickness).
In a similar manner to a procedure from [33], for cryo-SEM, insect samples were either glued with Tissue-Tek ® O.C.T.TM Compound (Sakura ® Finetek Europe B.V., Zoeterwoude, Netherlands) to metal holders or mechanically gripped in a small vice on holders and then frozen in a cryo-stage preparation chamber at −140 °C (Gatan ALTO2500 cryo-preparation system, Gatan Inc., Abingdon, UK). Frozen samples, either whole or fractured with a cold metal fracture knife in the cryostage preparation chamber, were sputter-coated with gold/palladium (6 nm thickness) and examined under freeze conditions (−120 °C) in the SEM Hitachi S-4800 at 3 kV acceleration voltage. Sputter-coated samples were also used to demonstrate the modulation of the light reflection properties of both black and white scales after introduction of a metal layer on top of the scales.

Transmission electron microscopy
In a similar manner to a procedure from [21], the tarsi and tibiae of Ae. albopictus were dissected from anaesthetized insects and fixed for 3 h in 2.5% glutaraldehyde in cacodylate buffer (Electron Microscopy Sciences, Hatfield, England), pH 7.2. The fixed legs were repeatedly rinsed in sodium cacodylate buffer and post-fixed for 1 h at 4 °C in 1% osmium tetroxide in sodium cacodylate buffer (Electron Microscopy Sciences). The samples were then repeatedly washed in the same buffer, dehydrated in ascending ethanol concentrations, and finally embedded in an Epon-Araldite resin mixture (Sigma-Aldrich). Afterwards, ultrathin sections were cut using a Leica EM UC6 ultramicrotome (Leica Microsystem GmbH, Wetzlar, Germany), collected on formvar-coated copper grids, and examined using a TEM Philips EM 208 (Philips, Eindhoven, Netherlands).

Reflectance spectra measurements
The light scattering spectra from the white areas on the proximal parts of the hind leg tarsus of Ae. albopictus male were measured at different orientations of the light source relative to the leg and at different detection angles. For this purpose, the observation area was illuminated at 45° with a light source (DH-2000-BAL, Ocean Optics Inc, Dunedin, Florida, USA) through an optical fiber (200 μm diameter) equipped with a one-lens condenser, which was placed 22 mm away from the sample. The illumination spot was one millimetre in diameter. The illuminated area was calculated based on the microscopy image of the leg (similar to Figure 1f). The mosquito hind leg was fixed using double-sided adhesive tape (Tesa ® , Hamburg, Germany) so that the tarsus was parallel to the horizontal plane with the dorsal side up. Measurements were performed for three different orientations of the tarsus relative to the light source, namely illumination along the tarsus main axis proximally and distally, and perpendicular to the tarsus main axis.
The light scattering was relatively homogeneous. Therefore, just three different angles were selected to characterize the reflection properties of the white areas. Reflection spectra at angle of incidence (45°, from the normal to the surface), smallangle scattering (30°), and the backscattering (−20°) were measured. The scattered light was collected by another condenser, which was placed 46 mm away from the sample and was mounted on an optical fiber (200 μm diameter), similarly to [18]. The optical fiber was connected to a spectrometer (Ocean Optics Inc, Dunedin, Florida, USA). The spectra were recorded with the software Spectral Suite (Ocean Optics Inc, Dunedin, Florida, USA). Further spectral processing was performed in Matlab 7.10 (The MathWorks, Natick, NA, USA) and includes dark noise subtraction and smoothing with a Savitzky-Golay filter of fourth order with 12 nm window. All spectra were normalized to the WS1 standard (Ocean Insight, Ostfildern, Germany) reflection spectrum (illumination and observation at 45°). No correction on the observation angle was performed.

Author Contributions
The study was designed by all the authors. S.G. performed the cryo-scanning electron microscopy investigations. M.R. and S.P. performed the transmission electron microscopy investigations. G.S. performed the light microscopy and the fluorescence microscopy investigations. A.K. performed the reflectance spectra observations. The manuscript was written by M.R., A.K. and S.G. All authors discussed the analysis and interpretation of the results and participated in the final editing of the manuscript.